Insulin–cobalt core–shell nanoparticles for receptor-targeted bioimaging and diabetic wound healing

Diabetic wounds represent a major issue in medical care and need advanced therapeutic and tissue imaging systems for better management. The utilization of nano-formulations involving proteins like insulin and metal ions plays significant roles in controlling wound outcomes by decreasing inflammation or reducing microbial load. This work reports the easy one-pot synthesis of extremely stable, biocompatible, and highly fluorescent insulin–cobalt core–shell nanoparticles (ICoNPs) with enhanced quantum yield for their highly specific receptor-targeted bioimaging and normal and diabetic wound healing in vitro (HEKa cell line). The particles were characterized using physicochemical properties, biocompatibility, and wound healing applications. FTIR bands at 670.35 cm−1, 849.79, and 973.73 indicating the Co–O bending, CoO–OH bond, and Co–OH bending, respectively, confirm the protein–metal interactions, which is further supported by the Raman spectra. In silico studies indicate the presence of cobalt binding sites on the insulin chain B at 8 GLY, 9 SER, and 10 HIS positions. The particles exhibit a magnificent loading efficiency of 89.48 ± 0.049% and excellent release properties (86.54 ± 2.15% within 24 h). Further, based on fluorescent properties, the recovery process can be monitored under an appropriate setup, and the binding of ICoNPs to insulin receptors was confirmed by bioimaging. This work helps synthesize effective therapeutics with numerous wound-healing promoting and monitoring applications.


Introduction
Wound healing is a complex process involving a sequential overlapping cascade of events that comes into action in response to some external chemical or physical stimuli and eventually causes healing by restoring lost tissue. 1 The healing process is categorized into four phases, hemostasis, inammation, proliferation, and remodeling. 2 The hemostasis is initiated by clot formation due to platelets' activation, which prevents microbial infestation and promotes matrix organization. In proliferation, the accumulation of cells, connective tissue, growth factors, and angiogenesis factors across the wound occurs. The remodeling involves the resynthesis of the extracellular matrix to maintain the balance between the death of existing cells and the formation of new cells. 3,4 However, the progress monitoring of recovery of wounds always remains a signicant challenge. In some instances, normal healing gets disrupted. It gets arrested in one of the phases due to the loss of balance in the physiological mechanism of healing due to infection, chronic irritation, trauma, the persistence of microbes or other foreign bodies, and ischemia, making the wound a chronic one 5-7 which can be prevented by keeping the wound moist, removing the dead tissues, covering the injury to avoid bacterial infection, and removing the excess tissue uid. 8 Apart from these issues, the major problem for delayed healing is diabetes mellitus or long-term hyperglycemia, which alters the secretion of cytokines, making wound healing cumbersome. 9 In diabetic conditions, there is the prolonged proinammatory phase due to the persistent release of proteases, proinammatory cytokines, and reactive oxygen species and the delayed anti-inammatory phase because of the low secretion of anti-inammatory cytokines. 10 Further, the transport of nutrients to the wound site is prevented by atherosclerosis caused by diabetes. 11 It also results in the dysfunction of endothelial cells due to vasodilation induced by pressure 12 and disrupts the processes essential for re-epithelialization; the proliferation of keratinocytes and broblasts, synthesis of proteins, and cell migration. [13][14][15] Further, it impairs the body's immune response, making the wounds prone to infection and leading to damaged structural components of the extracellular matrix. 7 It also causes free radical damage due to the reduced activity of certain antioxidant enzymes such as glutathione peroxidase and superoxide dismutase. 16 Various advanced bandages are developed for treating diabetic wound healing and overcoming the issues faced in effective healing. 17,18 Recent advancements in nanotechnology, such as specic physicochemical properties and small size, allow the intracellular delivery of different drugs and biomolecules, protect them from degradation, increase the penetration ability of drugs into the wound, allow the topical application, and enhance the halflife of this agents. 19,20 It results in a decline in repetitive drug application, thus lowering the cost and making them costeffective. 21,22 The metallic nanoparticles possess a high surfaceto-volume ratio, high stability, biocompatibility, safety, and economical rates. 22,23 Several gold, silver, copper, zinc, and cobalt metallic nanoparticles were prepared to study wound healing activities in vitro and in vivo models. 24 Protein-based nanoparticles are gaining attention due to their high biodegradability, easily metabolizable nature, and the possibility to alter or modify their surface for the drugs to attach efficiently. The proteins can be of plant or animal origin, such as bovine serum albumin, 25 insulin, 26 transferrin, 27 lactoferrin, and so on, for nano-formulation synthesis. 28,29 The selection of nanoparticles in treating wounds is based on three parameters; antimicrobial action, role as a delivery agent, and role in the repair process. 30 The use of insulin protein is increasing due to the enormous presence of insulin receptors on the membranes of all mammalian cells, and there is a signicant variation in the number of receptors from just 40 in erythrocytes to 200-300 × 10 3 for adipocytes and hepatocytes; however, the number is much higher in cancerous cells compared to normal cells. 26,31 It plays a crucial role as a growth factor that facilitates chemotaxis and pinocytosis or phagocytosis by macrophages, promotes the secretion of cytokines critical for inammation, and helps in reepithelialization, essential for wound healing. It works by transitioning proinammatory cytokines into anti-inammatory cytokines and promotes wound repair and regeneration. 32,33 The nano-formulations involving insulin as a protein template exhibit promising applications in bioimaging, super-resolution microscopy, normal wound healing, and diabetic wound healing. [32][33][34][35] The metal ions used earlier to form different formulations with insulin include silver, copper, zinc, nickel, etc. 31 Apart from metal-insulin nanoparticles, other nano-formulations involving insulin as a wound-healing agent have been synthesized. Insulin-containing chitosan nanoparticles were synthesized to stimulate inammatory cell proliferation, angiogenesis, and wound healing in diabetic rats. 36 Lee et al. reported accelerated diabetic wound repair by promoting epithelialization using core-shell insulin-loaded scaffolds. 37 The synthesis of human keratin conjugated insulin was reported by Li et al. for their role in full-thickness wound repair in rats by promoting cellular migration. 38 Clinical studies were also performed to conrm the role of insulin further. The insulin injection was used to study the effect on systemic blood glucose levels and healing of diabetic foot ulcers in patients and found that insulin promotes the formation of vessels and granulation tissue. 39 The impact of topical insulin was studied in 110 patients having diabetic foot ulcers using a double-blind placebo-controlled trial and found that patients treated with insulin gauze dressings had a wound diameter of 2.46 ± 0.57 cm aer two weeks, while patients treated with saline gauze had a diameter of 3.90 ± 0.76 cm indicating the signicant effect of insulin. 40 Similarly, a mucoadhesive liposomal gel of insulin was explored on patients with chronic wounds in different body parts, demonstrating a more signicant impact on test groups than control groups. 41 The cobalt ions also help in wound healing and act as an alternative to hypoxia-mimicking agents. It inhibits the activity of PHDs (prolyl 4-hydroxylases), articially stabilizes the HIF-1a (hypoxia-inducible factor-1a) under normal conditions, and activates the HIF pathway, 42 allowing the formation of the HIF complex, which gets translocated to the nucleus and causes upregulation in the expression of genes such as VEGF, which are responsible for adaptation to hypoxia. 43 As a result, it promotes angiogenesis during the tissue regeneration process. 44 It was also found that incorporating cobalt ions in titanium-based bone implants enhances their antibacterial activity, indicating the role of cobalt as an antimicrobial agent. 45 Further, the release of cobalt ions from the bioactive glass increases the VEGF protein secretion and osteogenic expression of genes by human bone marrow stromal cells. 46,47 Moreover, if the cobalt ions are delivered as cobalt chloride, they enhance the secretion of VEGF by endothelial cells, 48 broblasts, 49 and human mesenchymal stem cells. 50 The synthesis of silicate glass bers for delivering the cobalt ions was done for their chronic wound healing activity by activating the HIF pathway and enhancing the expression of angiogenic genes. 51 Similarly, a bioactive glass was made using the sol-gel method and doped with two different metal ions, including silver and cobalt, for their antibacterial activity in the case of wound healing applications. 52 In this work, we are reporting the synthesis of novel proteincoated metal core-shell nanoparticles for their in vitro normal and diabetic wound healing and bioimaging application for the rst time. Here, by using one-pot synthesis, we have prepared insulin-cobalt core-shell nanoparticles to check the synergistic effect of insulin and cobalt for treating wounds when applied in the form of particles in the nano range. The effective and efficient healing of wounds is essential for maintaining a person's life quality. Still, in some instances, the absence of proper internal wound monitoring makes the process even more complex, increasing the cost of treatment and putting risk to a patient's life. To overcome such challenges, the utilization of developed nanoparticles with dual healing and bioimaging capabilities offers a promising solution for enhanced wound management.

Synthesis of insulin-protected cobalt nanoparticles
At rst, the insulin-protected core-shell cobalt nanoparticles were prepared by following a previously reported one-pot method in which the pH of the insulin solution having a nal concentration of 1.82 mM, was adjusted to 10.5 using a NaOH solution (0.1 N) and kept in the dark. Aerward, a salt solution (CoCl 2 $6H 2 O) of the same molarity was prepared, followed by mixing two solutions in 1 : 1 by volume, which was further followed by adjustment of the pH of the nal solution to a physiological pH of 7.4, using HCl (0.1 N). The resulting solution was kept in the incubator at slow stirring (240 rpm) for 24 h at 37°C . [53][54][55] The nal solution was dialyzed using a 10 kDa cut-off dialysis membrane, stored at 4°C, and used further for characterization. Thus, produced insulin-cobalt nanoparticles were termed as ICoNPs.

Study of particle size, morphology, and elemental analysis
The DLS (dynamic light scattering) was done to determine the hydrodynamic size of ICoNPs formed using a Malvern DLS-Zeta size analyzer. Thereaer, to nd out the morphology of insulinlinked cobalt nanoparticles, Scanning Electron Microscopy (SEM JEOL, JSM-6300) and High-Resolution Transmission Electron Microscopy (HRTEM) (Talos F200S G2, Thermo Scientic) were used. For this, the samples were centrifuged at 240 rpm for 15 minutes, then thoroughly washed the pellet to remove unbound metal salt or impurities associated with the sample. Furthermore, the sample pellet was investigated to determine the percentage of the elements present by the Energy dispersive X-ray Spectrometer (EDS) (Bruker QUANTAX 200).

In silico studies
To determine the potential binding sites for different transition metal ions with the desired protein, MIB (Metal Ion Binding), an online docking tool, can be used as a user-friendly and integrated approach for nding the residues in metal binding sites. For operating this online server, protein chains were required, which were extracted from the protein data bank (PDB). The rst chain consists of the template protein (T) of length m containing metal-ion, and the second chain consists of query protein (S) of length n. These chains were then aligned properly so the metal ion binding protein template could be converted into the query protein structure aer alignment. Several different parameters were followed to obtain the desired protein structures. The rst and the most crucial parameter is the prediction of binding residues with the twelve transition metal ions, including Ni 2+ , Cu 2+ , Mg 2+ , Ca 2+ , Hg 2+ , Cd 2+ , Co 2+ , Zn 2+ , Fe 2+ , Mn 2+ , Cu + , and Fe 3+ . The second factor is that the polypeptide chain length in the protein structures should have 50 residues; otherwise, it will be excluded. 56 The third parameter is the presence of at least two metal ion binding residues having the minimum possible distance between the PDB coordinates and the metal center. 57 The fourth crucial parameter is the allotment of a binding score to the residue, which should be greater than a specied threshold value assigned and calculated using the root mean square deviation of C-alpha carbons of local structural alignment and BLO-SUM62 substitution. If so, only then will there be binding between the residue and the particular transition metal ion. Further, the binding score is denoted as CI and depends upon the target protein's sequence along with the protein's structural conservation. 58,59 Hence, the binding site of metal ions was determined using the bioinformatics tool.
In this manner, the binding site of a particular metal with that of a protein chain can be determined using the MIB tool. The PDB (Protein Data Bank) database was used to extract human insulin protein (PDB ID: 4EWW), a polypeptide with two distinct chains denoted as A and B, respectively. In the MIB tool, the Co 2+ metal ion was independently docked with chains A and B of human insulin, followed by a comparison of the metal ionbinding score with that of the target protein. 60

Study of cobalt-insulin interaction using spectroscopic techniques
To study the metal-protein interactions in the ICoNPs, FTIR was performed using an Agilent Cary 600 series Spectrophotometer. The potassium bromide (KBr) method was used to prepare the samples, and scanning was done from 400 cm −1 to 4000 cm −1 .
Furthermore, the same samples were analyzed using Surface Enhanced Raman Scattering (SERS) Spectra to study the structural changes in insulin protein. 63 All the samples were prepared on a silicon wafer ten minutes before the measurement. The samples were scanned from 500 cm −1 to 1800 cm −1 . The LabRam Hr Evolution Horiba, equipped with a detector and microscope, was used to record the Raman spectra of insulin and ICoNPs at 785 nm.
In order to monitor the changes in the secondary structure of the protein molecule, CD (Circular Dichroism) is the most reliable technique. In addition to the above studies, CD studies were conducted to conrm the interaction between insulin and cobalt chloride salt solution (freshly prepared and aer 1 month). We have performed the Circular Dichroism (CD) spectroscopy using the instrument Mos500 CD biologic. The CD studies were performed at 25°C with 1 ml of sample and scanned in the 200-260 nm wavelength range for pure insulin and insulin cobalt nanoparticles using phosphate buffer (pH 7.4) as a solvent.

Drug-loading and release kinetics
To monitor the release kinetics, 1 ml of the ICoNPs was centrifuged for 15 min at 10 000 rpm. The Bradford reagent determined insulin concentrations from the supernatant and the pellet. Further, the insulin release rate was measured at specic time intervals for 40 hours. The absorption values (at 595 nm) were later plotted to note the trend of drug release using BSA standard curves. 61,62 In addition, absorbance values were also measured for the same samples at 272 nm (excitation of tryptophan) to support the data further.

Absorption and uorescence spectroscopic study
For obtaining the UV-visible absorbance, the UV-2600 spectrophotometer of Shimadzu was operated between 200-800 nm, and a 4000 ml quartz cuvette with a 1 cm path was used, and the absorbance for insulin, salt solution (CoCl 2 ), and insulin linked cobalt core-shell nanoparticles (ICoNPs) were measured.
For measuring the uorescence data of ICoNPs, Agilent technologies, a Cary Eclipse uorescence spectrophotometer was used, which helps in determining the uorescence intensity of protein-linked metal nanoparticles (ICoNPs), insulin, and CoCl 2 solution, all having the same concentrations, at an excitation wavelength of 272 nm, coupled with an emission scan from 280 nm to 800 nm with excitation and emission slit of 10 mm.
In order to calculate and compare the percentage increase in intrinsic uorescence intensity of both insulin and ICoNPs, the given eqn (a) was used. The standard tyrosine uorescence quantum yield was used to calculate the quantum yield of insulin as well as insulinprotected cobalt nanoparticles (ICoNPs), and the given eqn (b) was used for further calculations. Here, Q.Y.is quantum yield; I is integrated emission intensity; n is the refractive index of solvent; A is the absorbance at excitation wavelength; l is the length of absorption cell; Tyr is tyrosine (reference), and S is sample.

Stoichiometry ratio of Co 2+ ions : insulin protein
To understand the interactions between metal-ligand complexes and complex proteins, the stoichiometry of cobalt ions to insulin protein is calculated using the following eqn (c).
No: of proteins bound to single Co 2þ ion ¼ Average no: of bound protein=ml Average no: of Co 2þ ions ml (c)

2.9
In vitro studies 2.9.1 Cell viability. In order to determine the viability of cells in the presence of all samples, the HEKa cell line (Human Epidermal Keratinocytes, adult) was used, and MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed. In order to perform this assay, HEKa cells were seeded into 96 well plates with a density of 1 × 10 4 cells per well and were kept in the incubator for the cells to grow and become 75-80% conuent with the regular addition of media. Once the plate became conuent, the cells were incubated with ICoNPs, insulin, CoCl 2, salt solution, and a mixture of insulin and CoCl 2 . To get three concordant readings, four different concentrations of each sample that are 1.5, 7.5, 30, and 60 mM are added to respective wells. Thereaer, the plate was kept in an incubator for a time-lapse of 24 h and at a temperature of 37°C. Aer 24 h, the media was discarded, followed by the addition of new media, and MTT (2 mg ml −1 in 5% ethanol) was added into each well, and the plate was placed as such for 3 h in an incubator. The solution of MTT and media was removed from each well aer 3 h, followed by the addition of 200 ml dimethyl sulfoxide (DMSO) to dissolve the formazan crystals. Finally, the absorbance was checked at a wavelength of 575 nm. To calculate the inhibition percentage, the equation used is as follows Here A t is the absorbance of the test substance, and A c is the absorbance of the control solvent for each concentration. 64 2.9.2 Internalization study of nanoparticles into cells. STEM analysis was performed to study synthesized nanoparticles' interaction with human cells. To do the internalization studies HEKa cell line was used. The cells were grown in 35 mm plates and let to be conuent till 80-85% and then were treated with ICoNPs and incubated for 24 hours. Aer 24 hours, the cells were trypsinized and centrifuged, and the pellet was xed using 2.5% glutaraldehyde in 0.03 M phosphate buffer having pH 7.4. Cells were then dehydrated using an ethanol gradient, later coated over 200-mesh uncoated copper grids, and observed under STEM (using the Carl Zeiss Sigma 500 Microscope).
2.9.3 Fluorescence bioimaging. To get the uorescence imaging, the Dewinter uorescence microscope was used. The cells were seeded in a 60 mm plate and let be conuent to 75-80%. Aer that, cells were placed on a coverslip, followed by adding DMEM media and incubated for 24 h. The samples to be used for bioimaging were kept under UV light in UV laminar for 30 minutes. Then, 100 ml of the insulin metal nanoparticles (ICoNPs) was taken from each of the three different concentrations described above for MTT assay and added to the respective coverslip containing HEKa cell line opted for the experiment that is, human epidermal keratinocytes, adults (HEKa). In order to get rid of any impurities, each coverslip was washed with PBS buffer twice, followed by cell xation by adding 2% of formaldehyde solution. Later on, the images were taken using the uorescence microscope once the cells were xed to check the role of insulin-protected cobalt core-shell nanoparticles and the effect of different concentrations of samples on uorescence intensity.
2.9.4 Effect of ICoNPs on recovery of the normal and diabetic wound, in vitro, using phase contrast imaging. To determine the effect of prepared nanoparticles on wound healing in vitro, the HEKa cells were seeded in 60 mm plates in the presence of glucose (360 mg dl −1 ) for diabetic wounds and in the absence of glucose for normal damages along with DMEM-F12 media (FBS-free medium). They were kept in an incubator at 37°C and 5% CO 2 level. The cells were allowed to grow and be 80-85% conuent. Once the plates became conuent, the cell scratch method was used to analyze the effect on healing. In this, the wound is created using a sharp object, that is, the sterile 200 ml tip, and was treated with different concentrations of ICoNPs, insulin, CoCl 2 salt solution, and the mixture of insulin and CoCl 2 salt (I + Co). Time-lapse imaging was done to nd the change in wound diameter, and the variation in the wound width was measured aer 6 h, 12 h, and 24 h, respectively. We randomly measured wound width at different positions for each scratch made in an individual well plate. We took the mean of those independent readings of wound diameter to calculate the percentage change in wound diameter for both the normal and diabetic wounds separately.

Determination of combination index (CI) for cobaltinsulin
The quantitative measure that provides the effect of a single drug in combination with another drug is Combination Index (CI). The level of synergism or antagonism is quantied by estimating the drug combination index in investigating synergistic or antagonistic drug combinations. The combination index of less than 1 (CI < 1) indicates that when different drugs are administered together, they work together to enhance each other's activity, called the synergistic effect. CI values equal to 1 (CI = 1) show that one drug does not interfere with the action of the other hence additive effect, and CI values greater than 1 (CI > 1) indicates the inhibition of drug action by the other drug, thus called the antagonistic effect. For the calculation of the combination index, the cell viability of HEKa cells was determined at varying concentrations of cobalt and insulin, and then calculations were done using eqn (e).

Statistical analysis
The data here is presented as the mean ± SD of at least three independent experiments. Using the one-way ANOVA, the statistical data analysis was done using MS Excel, and the corresponding p-value was calculated to check if the data was statistically signicant.

Structure, composition, and stoichiometry of metal insulin nanoparticles
The formation of spherical nanoparticles of ICoNPs having size 13 ± 2 nm was conrmed by scanning electron microscopic images (at a scale of 500 nm) as shown in Fig. 1a (inset shows the magnied image of core-shell particle which shows the rough surface of nanoparticle due to an outer insulin shell) and transmission electron microscopic images (at a scale of 50 nm) as shown in Fig. 1b and inset of Fig. 1b (at a scale of 5 nm to show the lattice fringes and the insulin coating of ∼2.5 nm around the cobalt core). The nanoparticles are widely distributed over the protein matrix. The presence of different elements like Co, C, O, N, P, and Si is shown in the energy-dispersive X-ray spectroscopy. The even distribution and percentage of Co in the particles come out to be 0.18% as analyzed by EDS studies as indicated in Fig. 1c. The hydrodynamic size of ICoNPs was found to be 35 ± 5 nm as shown in Fig. 1d. Thereaer, the stoichiometric ratio between the insulin protein and Co 2+ ions was calculated. It was found that ∼3792 insulin proteins encapsulate one nanoparticle. The detailed calculation is provided in ESI. †

In silico studies to monitor the interactions between insulin and cobalt ions using metal-ion binding residue templates
An online docking server determined the interaction sites between protein and metal ions, MIB (Metal Ion Binding). With this tool's help, we found the binding sites on chain B of insulin with which the transition metal Co 2+ can bind. The interaction of the protein with metals is based mainly on the structure and sequence of the amino acids in a protein. Firstly, the sequence of human insulin was extracted from the Protein Data Base (PDB ID: 4EWW) and then inserted in the MIB (Metal Ion Binding) tool to dock the Co 2+ ions with both the chains (chain A & B) of insulin. As a result, a binding score was assigned to each residue in the insulin protein. If the binding score exceeds the threshold, that particular residue is considered the binding site for that specic metal ion. Therefore, the binding sites for metal ions Co 2+ were determined. The data indicates that chain A does not show any template indicating the absence of binding sites of Co 2+ ions with chain A, whereas, in chain B, a binding site was found for Co 2+ ions. Then we worked with another soware called Maestro. Using this, we calculated the distance between the metal ion and its binding site, that is, amino acids, namely 8 GLY, 9 SER, and 10 HIS, present on chain B of insulin. The distance was found to be 1.69, 3.07, and 3.27, respectively. The binding score was also determined using MIB with different amino acids in chain B of insulin with Co 2+ ions, thus nding the best possible sites for attachment. The binding of cobalt ions with different amino acids is determined and visualized through different orientations of the metal-binding amino acids in proteins. The possible binding sites of cobalt ions with different amino acids in insulin are shown in Fig. 2a and b, the binding score of amino acids with insulin protein is shown in Fig. 2c, and the binding score values are given in Table  S1. †

FTIR, Raman spectra, and CD spectroscopy for the interactions between protein and metal ions aer the formation of nanoparticles
To gure out the interactions involved in metal-protein binding, FTIR was used for CoCl 2 , insulin, and ICoNPs. The peaks appeared due to intermolecular interactions of insulin and CoCl 2 at different wavenumbers. Firstly, the peak appeared at 506.70 cm −1 , which indicates the S-S stretching in insulin and ICoNPs. 32 Then a peak at 540. 25 Fig. 2d. Similarly, Raman spectral analysis was used to study the signicant conformational changes in insulin aer binding with cobalt ions. The S-S stretch and O-C-N bend were observed at 553.29 cm −1 and 786.44 cm −1 , respectively, in both insulin and ICoNPs. 32,72 The CoO-OH and Co-OH bond was observed at 895.84 cm −1 and 984.75 cm −1 , respectively, only in ICoNPs but was absent in insulin. 67  at 1372.70 cm −1 . 32 The peak that is responsible for amide-III random coils shis slightly from 1315.63 cm −1 in insulin to 1332.38 cm −1 in ICoNPs. 73 However, no such change was observed for amide-III (a-helix), as the peak appears at 1201.87 cm −1 in insulin and ICoNPs. 74 A slight shi was observed for amide-II from 1447.14 cm −1 in insulin to 1460.88 cm −1 in ICoNPs. 75 The peak for amide-I was observed at 1658.91 cm −1 and 1668.54 cm −1 , respectively, in insulin and ICoNPs. 76 The peak for the C]O stretch remains unchanged and is found at 1750.73 cm −1 in insulin and ICoNPs, 32 and this is shown in Fig. 2e. A comparative data of FTIR and Raman spectra are shown in Table 1.
Circular dichroism was performed using insulin protein and ICoNPs (freshly prepared and aer 1 month) to monitor the interactions between protein and salt solution and nd the stability of the secondary structure of protein aer binding with cobalt chloride. The three signicant far UV signals were recorded in both the solutions, including a positive peak at ∼200 nm and two negative peaks at ∼208 nm (a-helix) and 218 nm (b sheet), which were indicative of the secondary protein structure. The % variation in the peak at 200 nm is 5.23% and 6.21%, at 208 nm is 3.56% and 4.24% and at 218 nm is 4.82% and 5.71% in freshly prepared ICoNPs and ICoNPs aer 1 month; respectively and conrmed the protein stability with no signicant structural changes were observed, as shown in Fig. 3a.

Drug loading and release kinetic studies
The drug loading capacity and release kinetics of ICoNPs were studied, and the encapsulation efficiency of insulin over the cobalt nanoparticles was found to be 89.48 ± 0.049%. The release kinetic studies were performed at physiological conditions that are pH of 7.4 and a physiological temperature of 37°C . Burst drug release was observed for the initial 8 h, followed by a sustained drug release aerward. Most of the drug was released by the 40 h. The particles were suspended in a dialysis membrane with a concentration of 3.47 mg ml −1 , and the OD values were observed at 595 nm aer particular intervals of time. It was found that 92.21 ± 0.03% of the drug was released from ICoNPs by the end of 40 hours, making it an efficient drug delivery system in wound healing applications and is shown in Fig. 3b and c. For further conrmation of the release kinetics of the particles, absorbance value was taken at 272 nm (tryptophan absorbance) for all the samples aer specic time intervals which shows the gradual increase in OD values with increasing time, indicating the sustained release of insulin from particles and is shown in Fig. S1a. † 3.5 Spectroscopic changes aer synthesis of core-shell nanoparticles using absorbance and uorescence spectra Aer synthesizing ICoNPs, its spectroscopic characterization was done to observe the absorption and uorescence intensity of the formed nanoparticles. We obtained a sharp peak for insulin without any added metal salts at 271.61 with an absorbance value of 0.129, whereas, aer incubating the insulin with metal salts for 48 h, an absorbance value of 0.245 was obtained for nanoparticles having a peak at 271.50 nm. This conrms the synthesis of ICoNPs, as shown in Fig. 3d. The absorption peak is also compared with tyrosine, as it was selected as a standard and demonstrated in Fig. S1b. † The ICoNPs were then excited at an excitation wavelength of 270 nm to determine the uorescence intensity. An emission peak ranging from 280-440 nm was obtained with maxima at ∼303 nm with an intensity of 304.89 a.u when monitored from 200-800 nm, as shown in Fig. 3e. The % change in intrinsic uorescence intensity was found to be 58.88%. Further, to determine the quantum yield of ICoNPs, tyrosine was used as a standard as its quantum yield value is known. The quantum yield of insulin was found to be 0.1798, and that of ICoNPs turned out to be 0.6825, indicating the uorescent behavior of formulations synthesized and is shown in Fig. S1c. †

3.6
In vitro cell studies 3.6.1 HEKa cell viability assay. A cell viability test was done to check whether the samples could be employed for biological purposes, which further depends on the mitochondrial activity of cells. Testing was done for insulin, CoCl 2 , a mixture of insulin and CoCl 2, and ICoNPs using 1.5, 7.5, 30, and 60 mM of concentrations, and the graph was plotted. The cell viability of untreated cells (control) is taken as 100, and the rest of the studies are compared to this. For CoCl 2 treated cells, cell viability comes out to be 96.54 ± 6.43%, 102.12 ± 6.36%, 106.75 ± 5.58%, and 110.99 ± 0.77% for 1.5, 7.5, 30, and 60 mM respectively. Insulin shows 99.65 ± 8.14% for 1.5 mM, 105.02 ± 1.48% for 7.5 mM, 110.99 ± 2.89% for 30 mM and 120.26 ± 7.70% for 60 mM respectively. The cells exposed to a combination of insulin and CoCl 2 showed more cell viability. It is 109.97 ± 2.30%, 108.32 ± 4.17%, 122.78 ± 0.28%, and 127.57 ± 0.56%, respectively, for 1.5, 7.5, 30, and 60 mM respectively. Cell viability aer treatment with core-shell ICoNPs showed much more signicant changes, which are 110.44 ± 0.70% for 1.5 mM, 117.83 ± 8.76% for 7.5 mM, 134.72 ± 1.76% for 30 mM and 144.69 ± 0.98% for 60 mM respectively and comparative data is shown in Table 2. The graph demonstrated that none of all four samples is toxic to the cells when used in the above-mentioned concentrations. Moreover, with increasing concentration of Table 1 The comparative wavenumber values indicated by FTIR (in the range 400-4000 cm −1 ) and Raman spectra (500-1800 cm −1 ), respectively, of insulin, CoCl 2, and ICoNPs showing the position of different functional groups present, which coins the interaction amongst insulin and CoCl 2 salt solution indicating the formation of insulin cobalt core-shell nanoparticles samples added, the cell viability enhanced (shown in Fig. 4) and was maximum for core-shell ICoNPs formed, indicating that nanoparticles formed are not at all toxic; instead, they help in the cell division and multiplication and thus can be used for wound healing.
3.6.2 STEM analysis for cellular internalization of ICoNPs. STEM analysis was performed aer incubating the cells with samples to conrm the interaction and internalization of ICoNPs into the skin cells, followed by their xation and dehydration by ethanol gradient. The cells were observed under the microscope on copper grids. The mechanism behind cellular internalization can be explained as follows. The synthesized nanoparticles comprise the core-shell structure with cobalt as the core covered by an outer insulin shell. This insulin layer is responsible for the interaction of nanoparticles with human cells. The plasma membrane of target cells in the human body has insulin receptors to which insulin binds and exerts the physiological effect. Insulin receptors have two insulin binding sites that exhibit negative co-operativity; that is, insulin binding to one place prevents its binding to the other and keeps them active throughout. Once the particles bind to the receptors, they can enter the cell membrane's lipid bilayer by following energy-independent direct transduction. Moreover, due to the structural similarity between insulin and IGF-I, it can also bind to IGF receptors and show anti-inammatory activity. Aer entering the cell, insulin plays a crucial role in wound healing by controlling distinct mechanisms, including glucose metabolism, protein biosynthesis, and lipid biosynthesis. The interaction of ICoNPs with cellular receptors and their internalization into the cell membrane is shown in Fig. 5a-d (at different scales).
3.6.3 Fluorescence microscope bioimaging. To conrm the broad applications of core-shell insulin-cobalt nanoparticles for cellular imaging, bioimaging was performed on HEKa cell lines. Cells were incubated with particles and showed bright blue uorescence due to the binding of ICoNPs with the insulin receptors present on the cell wall of each cell, and the uorescence further goes on increasing with an increase in the concentrations of ICoNPs, that is, 7.5 mM, 30 mM, and 60 mM respectively. It suggests the nanoparticles illuminate the cell walls with bright blue color; therefore, they can be used for bioimaging to monitor the changes inside the cells during the entire healing process.
3.6.4 HEKa cell migration assay in diabetic and normal wound conditions. The core-shell metal protein nanoparticles induce cell migration aer treatment with CoCl 2 , insulin, insulin + CoCl 2 mixture, and ICoNPs in normal and diabetic wounds. However, the increase is more in normal injuries than in diabetic wounds. With increasing time, the extent of cell division and migration increased when monitored at a xed concentration of 30 mM. For the change in wound diameter, we measured wound width at three distinguished positions for each scratch made on an individual plate. We took the mean of those independent readings of wound diameter to calculate the percentage change in wound diameter with time. The change in wound diameter of HEKa cells taken as control aer 0, 6, 12, and 24 h is indicated in Fig. 6(a)a-d. In diabetic conditions, the cells treated with core-shell ICoNPs show the percentage change of the scratched wound aer 6, 12, and 24 h as 41.4 ± 1.08%, 54.94 ± 0.97%, and 67.66 ± 0.28%, respectively, and is shown in Fig. 6(a)q-t. A similar procedure was followed, and the % of a gap le in cells that were treated with CoCl 2 , insulin, and insulin + CoCl 2 was calculated and showed signicant migration when compared to control but less than that of core-shell ICoNPs. The % change in a migration aer 6, 12, and 24 h is noticed in all samples and came out to be 27.12 ± 0.50%, 45.58 ± 1.26%, and 54.45 ± 0.35% in cells incubated with a mixture of insulin + CoCl 2 ( Fig. 6(a)m-p). In cells treated with insulin, the value of change in migration is 20.27 ± 0.35%, 37.08 ± 0.35%, and 43.29 ± 0.33%, respectively ( Fig. 6(a)i-l), while it is 7.88 ± 0.60%, 21.09 ± 0.35% and 24.83 ± 0.70% in cells treated with CoCl 2 (Fig. 6(a)e-h). A comparative study of variation in scratch diameter of the diabetic wound aer treating the cells with all four samples is shown graphically in Fig. 6(a)u. Table 2 The table shows the % variation in mitochondrial reductase activity in the MTT assay for determining cellular metabolism rate using HEKa cells. The cells were treated with varying concentrations of control (CoCl 2, insulin, mixture of CoCl 2 and insulin) and ICoNPs, that is, 1.5 mM, 7.5 mM, 30 mM, and 60 mM respectively, for a duration of 24 hours. The data were plotted as mean value ± SD of three independent experiments. The combination index (CI) values for the cell viability for varying combinations of cobalt salt with insulin protein were calculated to check if the two drugs are synergistic or antagonistic, and the values come out to be less than 1, indicating the synergistic effect of drugs  Similarly, in the normal wound, the variation in scratch diameter for control cells is shown in Fig. 6(b)a-d. The % change in wound diameter is monitored aer the same time interval in normal conditions and found to be 14.84 ± 0.60%, 31.75 ± 0.35%, and 34.7 ± 0.70% in the cells treated with CoCl 2 (Fig. 6(b)e-h). In the cells treated with insulin, insulin + CoCl 2, the % variation in migration with time is more as compared to CoCl 2 and is equal to 30.33 ± 0.80%, 41.27 ± 0.70%, and 48.98 ± 0.35% in insulin ( Fig. 6(b)i-l), 40.65 ± 0.35%, 50.8 ± 0.35% and 59.19 ± 0.35% in insulin + CoCl 2 ( Fig. 6(b)m-p). The coreshell ICoNPs show the maximum migration, and the % wound diameter changed is 56.13 ± 0.92%, 63.50 ± 0.70%, and 71.43 ± 0.35%, respectively, indicating the signicance of prepared nanoparticles in the healing application of normal wounds ( Fig. 6(b)q-t). The graph in Fig. 6(b)u shows the variation in normal wound diameter as a comparative study of all the formulations aer particular time intervals. A comparative data table of change in wound migration in normal and diabetic wounds is shown in Table S2. † The statistical signicance of data was determined by calculating p values for the scratch assay in diabetic and normal wound conditions using one-way ANOVA. The comparative data are presented in Table S3. †

Combination index of cobalt and insulin
To calculate CI, Dm was calculated using m and y from Fig. S2a † for cobalt and Fig. S2b † for insulin. It was found from the CI's calculated values that the cobalt chloride salt and insulin show a synergistic effect. The values calculated for varying combinations of insulin and cobalt chloride concentrations come out to be less than one indicating the synergism between the two and, thus, work together to enhance each other's activity. This is in accordance with the results obtained for cell viability and cell migration assay indicating the signicant role of insulin cobalt nanoparticles in faster wound healing applications. The data is shown in Table 2.

Conclusion
The wounds were sometimes reopened to maintain the proper healing environment and eliminate any complications in the wound recovery process, paving the way for improper healing, enhanced healthcare costs, and decreased life quality. Thus, suitable agents are always required, which assist in selectively binding to the wound site and guide in monitoring the progress of wound recovery. We formulated novel core-shell ICoNPs containing insulin and cobalt and evaluated their role in wound healing activity. Insulin promotes cellular growth by modulating inammation, reduces blood glucose concentration, and exhibits anti-inammatory activity. Cobalt is known to reduce the microbial contamination load. Thus, the formulations were checked for a synergistic effect in wound treatment. Different techniques were used to conrm their formation, including TEM micrography, elemental analysis, FTIR, Raman spectroscopy, and metal ion binding site prediction and docking server (MIB) to ensure Co-metal interaction with the insulin protein.
From FTIR and Raman spectra, we got peaks showing the intermolecular interaction between Co and insulin and exhibiting moderate structural changes in amide regions of the protein. The metal-ion binding site prediction and docking server were used to nd possible sites for the metal ion to bind with insulin amino acid chains A and B. No site was observed in chain A, while in chain B, there was a binding site for Co 2+ ions, and the amino acids involved in groove formation were 8 GLY, 9 SER, and 10 HIS. The loading efficiency and release kinetic studies were done to determine the effective loading of insulin drugs over the nanoparticles. The synthesized core-shell nanoparticles were tested for their effect on cell viability to be used further for diabetic and normal wound healing and bioimaging applications. The calculated values for the MTT assay and combination index values indicate the synergism between the cobalt chloride salt and insulin protein, making them suitable for further utilization in biological activities. The formed core-shell nanoparticles, ICoNPs, show excellent target-specicity. Due to the insulin consumption in their synthesis, they exhibit the property of binding to insulin receptors, making these particles unique from other particles in various respects, mainly in target-specicity properties. Further, they have enhanced uorescent properties when compared with insulin alone which can be easily detected using uorescence microscopy and uorescence bioimaging. They also possess an enormous tendency to heal both diabetic and normal wounds. Thus, it has a wide range of applications in healing, bioimaging, and material and biomaterial sciences. Therefore, it can be further explored for in vivo applications in diverse elds.

Conflicts of interest
Authors declare no conict of interest.